The process of blood coagulation, thrombosis, is triggered by a complex proteolytic cascade leading to the formation of thrombin. Thrombin proteolytically removes activation peptides from the A.alpha. and B.beta.-chains of fibrinogen, which is soluble in blood plasma, initiating insoluble fibrin formation.
Anti-coagulation is currently achieved by the administration of heparins and coumarins. Parenteral pharmacological control of coagulation and thrombosis is based on inhibition of thrombin through the use of heparins. Heparins act indirectly on thrombin by accelerating the inhibitory effect of endogenous antithrombin III (the main physiological inhibitor of thrombin). Because antithrombin III levels vary in plasma and because surface-bound thrombin seems resistant to this indirect mechanism, heparins can be an ineffective treatment. Because certain coagulation assays are believed to be associated with efficacy and safety, heparin levels are typically monitored with coagulation assays (particularly the activated partial thromboplastin time (APTT) assay). Coumarins impede the generation of thrombin by blocking the posttranslational gamma-carboxylation in the synthesis of prothrombin and other proteins of this type. Because of their mechanism of action, the effect of coumarins can only develop slowly, 6-24 hours after-administration. Further, they are non selective anticoagulants. Coumarins also require monitoring with coagulation assays (particularly the prothrombin time assay).
To better understand the invention, the following brief description of the coagulation enzyme system is provided. The coagulation system, sometimes referred to as the "cascade", is best looked at as a chain reaction involving the sequential activation of zymogens into active serine proteases which eventually lead to the production of the enzyme, thrombin. Thrombin, through limited proteolysis, converts plasma fibrinogen into the insoluble gel, fibrin. Two key events in the coagulation cascade are the conversion of clotting Factor X to Xa by clotting factor IXa and the conversion of prothrombin into thrombin by clotting factor Xa.
Both of these reactions occur on cell surfaces, most notably the platelet endothelial cell surfaces, and both reactions require cofactors. The major cofactors, factors V and VIII, circulate as relatively inactive precursors, but when the first few molecules of thrombin are formed, thrombin activates, by limited proteolysis, the cofactors. The activated cofactors, Va and VIIIa, accelerate, by about three orders of magnitude, both the conversion of prothrombin into thrombin and the conversion of factor X to factor Xa.
Activated protein C overwhelmingly prefers two plasma protein substrates which it hydrolyzes and irreversibly destroys. These plasma protein substrates are the activated forms of clotting cofactors V and VIII (cofactors Va and VIIIa, respectively). Activated protein C only minimally degrades the inactive precursors, clotting factors V and VIII. In dogs, activated protein C has been shown to sharply increase circulating levels of the major physiological fibrinolytic enzyme, tissue plasminogen activator.
The activation of protein C, however, involves thrombin, the final serine protease in the coagulation cascade, and an endothelial cell membrane-associated glycoprotein, thrombomodulin. Thrombomodulin forms a tight 1:1 stoichiometric complex with thrombin. Thrombomodulin, when complexed with thrombin, modifies substantially the functional properties of thrombin. Thrombin, in the coagulation pathway, normally clots fibrinogen, activates platelets, and converts clotting cofactors V and VIII to their activated forms, Va and VIIIa. Thrombin, alone, acts to activate Protein C, but only very slowly and inefficiently. In contrast, thrombin, when in the a 1:1 complex with thrombomodulin, fails to clot fibrinogen, does not activate platelets, and does not convert clotting factors V and VIII to their activated forms. The thrombin:thrombomodulin complex promotes the activation of protein C with the rate constant of protein C activation being as great as 20,000-fold higher for the thrombin:thrombomodulin complex than the rate constant for thrombin alone.
Activated protein C, therefore, is an antithrombotic agent with a wider therapeutic index than other anticoagulants, such as heparin and the oral hydroxycoumarin-type anticoagulants, such as warfarin. Neither protein C nor activated protein C is effective until thrombin is generated at some local site. Activated protein C is virtually ineffective without thrombin, because thrombin is needed to convert clotting factors V to Va and VIII to VIIIa. As noted, the activated forms of these two cofactors are the preferred substrate for activated protein C. The protein C zymogen, when infused into patients, will remain inactive until thrombin is generated. Without the thrombomodulin:thrombin complex, the protein C zymogen is converted into activated Protein C at a very slow rate.
The present invention is directed to the discovery that the compounds of the present invention, as defined below, increase thrombomodulin expressions and have oral bioavailability.